A separation chamber to sort cells, nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells
✍ Scribed by A. Tulp; M.W. Kooi; J.B.A. Kipp; M.G. Barnhoorn; F. Polak
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 983 KB
- Volume
- 117
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A separation chamber having a surface of 50 cm* and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speedcontrolled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 2Og, (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.